Rescuing Self: Transient Isolation and Autologous Transplantation of Bone Marrow Mitigates Radiation-Induced Hematopoietic Syndrome and Mortality in Mice

Abstract

The inflamed bone marrow niche shortly after total body irradiation (TBI) is known to contribute to loss of hematopoietic stem cells in terms of their number and function. In this study, autologous bone marrow transfer (AL-BMT) was evaluated as a strategy for mitigating hematopoietic form of the acute radiation syndrome by timing the collection phase (2 h after irradiation) and reinfusion (24 h after irradiation) using mice as a model system. Collection of bone marrow (BM) cells (0.5 × 10⁶ total marrow cells) 2 h after lethal TBI rescued different subclasses of hematopoietic stem and progenitor cells (HSPCs) from the detrimental inflammatory and damaging milieu in vivo. Cryopreservation of collected graft and its reinfusion 24 h after TBI significantly rescued mice from lethal effects of irradiation (65% survival against 0% in TBI group on day 30th) and hematopoietic depression. Transient hypometabolic state (HMS) induced 2 h after TBI effectively preserved the functional status of HSPCs and improved hematopoietic recovery even when BM was collected 8 h after TBI. Homing studies suggested that AL-BMT yielded similar percentages for different subsets of HSPCs when compared to syngeneic bone marrow transfer. The results suggest that the timing of collection, and reinfusion of graft is crucial for the success of AL-BMT.

Keywords: autologous bone marrow transfer; hematopoietic stem cells; hematopoietic syndrome; homing; ionizing radiation; mitigation.

Figure 1

Bone marrow collected within 2 h after total body irradiation (TBI) rescues mice from lethal effects when reinfused 24 h later. (A) Schema of autologous bone marrow transplantation setup. Bone marrow was collected 2 h after TBI (8.5 Gy), cryopreserved for 22 h, and reinfused 24 h after TBI. (B). 2 h after TBI, SJNP-1 was administered through i.p. route, and mice were kept at an ambient temperature (T_a) of 15°C for 6 h. Thereafter the mice were shifted to T_a (25°C) for recovery and 2 h later [that is, 8 h after hypometabolic state (HMS)] bone marrow was collected, cryopreserved, and reinfused 24 h after TBI. (C). Kaplan–Meier plot showing survival rates of mice with different treatments.

Figure 2

Effect of bone marrow collection time on radiomitigative effect of AL-BMT. (A) Schema of autologous bone marrow transplantation. Bone marrow was collected at indicated time points after total body irradiation (TBI) (8.5 Gy), cryopreserved and reinfused at 24 h after TBI. (B). Kaplan–Meier plot showing survival rates of mice with different treatments.

Figure 3

Hematopoietic parameters of recipient mice transplanted with autologous bone marrow collected 2 h after lethal irradiation or 8 h after induction of hypometabolic state (HMS). Changes in the number of white blood cell (WBC) (A), lymphocytes (B), granulocytes (C), RBC (D), hemoglobin (E), and platelets (F) measured on 30th postirradiation day. Each value is a mean ± SEM (n = 4–6 animals/group), and comparisons were done as indicated using unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant).

Figure 4

Cryopreservation procedure preserves different hematopoietic stem and progenitor cells (HSPCs) better than in irradiated animals in vivo. (A) Schema of experimental setup. (B) Bone marrow was aspirated 2 h after lethal total body irradiation (TBI), cryopreserved for 22 h followed by processing and enumeration of different subsets of HSPCs. Bone marrow collected 24 h after TBI or from vehicle-treated un-irradiated animals were used for comparison. The fractions of different HSPCs that are annexin V positive were deducted from their total number and used for comparisons. Each value is a mean ± SEM (n = 4–6 animals/group), and comparisons, as indicated, were done for statistical significance using unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Homing of different hematopoietic stem and progenitor cells (HSPCs) in autologous and syngeneic BMT setup. (A) Schema of experimental setup for evaluating homing of different HSPCs in case of AL-BMT (i), SG-BMT with surgery (ii), and SG-BMT without surgery (iii) setup. (B–G) Number of different subsets of HSPCs homed into femur 24 h after transplantation of indicated grafts with or without surgical manipulations. Each value is a mean ± SEM (n = 4 animals/group), and comparisons, as indicated, were done for statistical significance using unpaired Student’s t-test (n = 4; *p < 0.05, **p < 0.01). Surg, surgery.

Figure 6

The proliferation status of transplanted bone marrow cells that have homed into bone marrow. (A) Representative example of carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay. Bone marrow was harvested 72 h after transplantation, and CFSE⁺ cells were gated and mean fluorescence of the population was analyzed for CFSE dilution. Undivided bone marrow cells (red), hematopoietic progenitor cells (HPCs) (black), KSL (green), short-term hematopoietic stem cells (ST-HSCs) (gray), and long-term hematopoietic stem cells (LT-HSCs) (blue) (n = 4). (B) Enumeration of different subsets of CFSE⁺ hematopoietic stem and progenitor cells (HSPCs) in bone marrow harvested 72 h after AL-BMT. Each value is a mean ± SEM (n = 4 animals/group), and comparisons, as indicated, were done for statistical significance using unpaired Student’s t-test (n = 4; *p < 0.05, **p < 0.01).